Hepatic cytochrome P-450 is known to be involved in the metabolism of steroids, drugs and other xenobiotics. Substrate specificity of this enzymatic system is determined by P-450 and the presence of multiple forms of P-450 in the hepatic microsomes is well documented. Our laboratory has been interested in the purification as well as the functions of a specific sex-dependent and neonatally imprinted form of P-450. Utilizing various column chromatography steps (DEAE, hydroxyapatite and octyl-amino-Sepharose), we have partially purified this form of hepatic cytochrome P-450 to a specific content of 7.5 nmoles/mg. The major hemoprotein in the purified preparations has an apparent molecular weight of 50,000 dalton as determined by SDS gel electrophoresis. In a reconstituted system, this form of P-450 is capable of hydroxylating testosterone at the 16 alpha-position with a turnover number of 1.4 which is about 10-fold that of the microsomal suspension. Cytochrome P-450 similarly purified from adult females or neonatal castrates failed to hydroxylate testosterone at the 16 alpha-position whereas adult rats castrated at 4 weeks exhibited the same 16 alpha-hydroxylase activity as that of the control males. These results suggest that neonatal androgen is responsible for the imprinting of a specific form of hepatic cytochrome P-450 which could account for the subsequent differences in substrated hydroxylation between neonatally imprinted and non-imprinted rats. We propose to explore further the substrate specificity and immunochemical properties of this form of hepatic cytochrome. Utilizing this approach, we have also studied the imprinting of hepatic glutathione transferase B. The immunochemical methods have been used to determine the nature of gene expression in hepatic cells.